Cellular membrane lipids serve as a source of lipid mediators which regulate the signaling mechanisms that govern mitogenesis. Since dietary fats provide lipids used for cellular membranes, fat intake could affect the quantity and quality of lipid mediators that are available for regulating mitogenic activity. Growth factors and hormones stimulate the production of lipid mediators by activating phospholipase D. This phospholipase hydrolyzes membrane phospholipids to provide the lipid mediators phosphatidic acid and diacylglycerol. The influence of dietary fat on the production of these lipid mediators and the regulation of phospholipase D is the focus of this work. A cell culture model using tumorigenic and nontumorigenic human breast epithelial cells has been established to study the effects of membrane lipids on cell signaling mechanisms. The cells respond with increased phospholipase D activity when exposed to a variety of physiologically relevant agonists including epidermal growth factor, transforming growth factor alpha and phorbol esters. The phospholipase D is differentially regulated in tumorigenic breast cells which are resistant to drugs commonly used for chemotherapy. The fatty acids of these cells can also be modified in a manner that simulates dietary changes. The characteristics of the phospholipase D and the ability to modify the membrane lipids are valuable assets of the model that will be used to study the effects of dietary fat on cell signaling mechanisms that regulate mitogenesis.